PMC

A26 and A27 form an SDS-stable complex. (A) Western blot analysis of virions. Virions from cells infected with VACV WR (vWR) and vΔA26 were purified by sucrose gradient sedimentation, dissociated with LDS in the presence or absence of reducing agent, and subjected to SDS-PAGE. The blots were probed with antibody to A26 and then stripped and reprobed with antibody to A27. The positions of monomeric and oligomeric forms of A27, monomeric A26, and A26/A27 complex are indicated. (B) Immunoaffinity purification of A26/A27 complex. Purified virions from cells infected with VACV WR or vA26V5 MV were treated with 1% NP-40 detergent and incubated with antibody to V5 or A27 immobilized on protein A beads. Bound (B) and unbound (U) fractions were resolved by SDS-PAGE under nonreducing conditions and probed successively with antibody to A27 and V5 (left) or with antibody to V5 (right). Pre, eluate from protein A-agarose beads unconjugated to antibody. (C) Western blot of cell lysates. Cells infected with VACV WR or vΔA26 were harvested after 24 h and lysed in the presence (+) or absence (−) of 50 mM NEM. The proteins were resolved by SDS-PAGE under nonreducing conditions, and Western blots were probed with antibody to A26 and A27. In each panel, the numbers on the left are the masses in kDa of marker proteins.

A26 is anchored to the MV membrane surface by A27. (A) Biotinylation of A26 and A27 in intact virions. Sucrose gradient-purified virions from cells infected with VACV WR were treated with sulfo-NHS-SS-biotin and allowed to bind to NeutrAvidin beads. Bound (B) and unbound (U) fractions were analyzed by Western blotting with antibodies that recognized A26, A27, and A3. As a positive control, purified virions were treated with NP-40 prior to biotinylation. (B) Incorporation of A26 into purified virions. Cells were infected with vΔA27 and transfected with pA26V5 with or without pA27. After approximately 24 h, the cells were harvested and virions were purified by sedimentation through sucrose cushions and CsCl gradient sedimentation. Fractions (1 ml) were collected from the top and analyzed by Western blotting using antibodies that recognized A26V5, A27, and A3.

Cysteines 71 and 72 of A27 protein are required for the formation of A26-A27 protein complexes. Schematic representation of the cysteine residues 71 and 72 on A27 protein. BSC40 cells were mock infected or infected with IA27L-A26WR and subsequently transfected with individual plasmids encoding wild-type A27 or C71A, C72A, or C71/72A double mutant proteins. Cells were harvested at 24 h p.i., boiled, and separated on nonreducing SDS-PAGE for immunoblot (IB) analyses using anti-A27 Ab (A) or anti-A26 Ab (B). The black and white arrows indicate the 90- and 70-kDa protein complexes, respectively. A27-m, A27-d, and A27-t represent monomers, dimers, and trimers of A27 protein, respectively. Tf, transfection.

The localization of GUS protein in the transgenic alfalfa plants in the absence of R. meliloti. A, Transverse section of a root of plant line A27 (MsENOD40–2). Blue color was detected in the stele, in the pericycle, endodermis, and inner cortex; bar = 55 μm. B, Cross-section of a stem of plant line A17 (MsENOD40–2). Blue color is localized to the stem procambium and the protophloem; same magnification as in C. C, Longitudinal section of a root tip of plant line A17 (MsENOD40–2). Note that the blue staining is present even in the root cap; bar = 110 μm. D, Lateral root primordium on transgenic plant line A27 (MsENOD40–2) same magnification as in C. E and F, Elongated lateral roots on the same plant shown in D; bar = 110 μm. G, Root of plant line a18 (MsENOD40–1) without any treatment; bar = 110 μm. H, Root of plant line a18 4 d after treatment with 10⁻⁶ m BAP. I, Root of plant line a18 4 d after treatment with 10⁻⁸ m PNF. J, Root of plant line A27 (MsENOD40–2) without any treatment. H through J are the same magnification as in G. K, A 10⁻⁶ m BAP-treated root of line A27 after 4 d; bar = 110 μm. L, Root of line A27 treated with 10⁻⁸ m PNF for 4 d. M, Root of line A23 treated with 10⁻⁶ m 2,4-D for 4 d; no blue color is present. L and M are the same magnification as in K. N, Nodule primordium formed on plant line a18 (MsENOD40–1) after BAP treatment for 4 d; bar = 55 μm. O, Cross-section of a primordium in N that was induced by BAP. P, Primordium formed on plant line A27 (MsENOD40–2) after BAP treatment for 4 d. Same magnification as N. Q, Nodule primordium formed on plant line A27 (MsENOD40–2) following 4 d of PNF treatment; bar = 55 μm. Arrows indicate starch grains and the arrowhead points to a root hair that is out of the plane of section.